Metformin Inhibits Growth Of Eutopic Stromal Cells From Adenomyotic Endometrium Via AMPK Activation And Subsequent Inhibition Of AKT Phosphorylation:a Possible Role In The Treatment Of Adenomyosis

Part IThe expression of protein AMPK in endometrial stromal cellsObjective1. To investigate the expression of AMPK in normal endometrial stromal cells (N-ESCs) and adenomyotic eutopic endometrial stromal cells (A-ESCs)2. To detect the effect of Compound C to the expression level of AMPK in N-ESCs and A-ESCs.3. To explore the conditions of Compound C for blocking AMPK pathway.Methods1. Paired fresh normal endometria and adenomyotic eutopic endometria were collected from untreated patients subjected to radical hysterectomy. Then endometrial stromal cells were got by primary cell culture.2. Immunocytochemistry (IHC) was used to detect the purity of the ESCs and the expression of AMPK in N-ESCs and A-ESCs.3. Western blot (WB) assay was used to detect the AMPK protein level in N-ESCs and A-ESCs.4. With or without Compound C, we use Western blot assay to detect the expession of AMPK protein level in N-ESCs and A-ESCs.Result1. The purity of the ESCs was98%after the first and second passages. 2. AMPK was positive expressed in the cytoplasm of both N-ESCs and A-ESCs; A-ESCs exhibited greater AMPK expression than N-ESCs (P<0.05); AMPK expression levels (AMPK/β-actin) in A-ESCs and N-ESCs14groups were83.4%±13.8%and58.9%±9.8%, respectively (i.e.1.4-fold more).3. Compound C was found to significantly inhibit the phosphorylation of AMPK in a time-dependent manner. In both N-ESCs and A-ESCs, after1h exposure of Compound C, the average inhibition was2.4±0.5-fold2in A-ESCs and2.9±0.9-fold in N-ESCs.ConclusionAMPK was positive expressed in the cytoplasm of both N-ESCs and A-ESCs; A-ESCs showed stronger expression level than N-ESCs. Compound C can block AMPK pathway. Part II:The effect and the possible mechanism of metformin on the growth of A-ESCsObjective1. To evaluate the effect of metformin on the growth of endometrial stromal cells (ESCs).2. To compare the effect of metformin on A-ESCs variation with the menstrual cycle 3. To explore the possible mechanisms of metformin on A-ESCsMethods1. The functional effect of metformin on cell proliferation was evaluated using MTT assay.2. The application of Western blotting (Western Blot) was used to detect the effect of metformin on AMPK activation.3. Western Blot and MTT assay were used to explore the different effect during menstrual cycle.4. Western Blot was used to detect the relationship between AMPK and Akt pathway.Results1. Metformin can inhibit endometrial stromal cell growth inhibition in a dose dependent manner.N-ESCs:IC507.87mmol/L; A-ESCs:IC502.45mmol/L.2. Metformin was associated with a significant2.0±0.3-fold increase in AMPK activation (p-AMPK/AMPK) compared with controls (*P<0.001); The relative activated AMPK levels in A-ESCs in control, metformin, compound C, and compound C+metformin treated groups were43.9%±1.6%,83.2%±.1%,23.2%±5.2%, and25.1%±4.1%,3. Metformin inhibited the growth of A-ESCs from proliferative phase by47.8%±1.2%and cells from secretory phase by58.0%±0.9%relative to controls. The effects of metformin on activating AMPK18signaling (p-AMPK/AMPK) were obviously(*P<0.001).The relative activated AMPK levels in cells during the secretory phase and proliferative phase1were86.8±4.8(2.3-fold increase versus control),60.2±2.5(1.6-fold increase versus2control), respectively.4. The inhibitory effects of metformin on AKT activation (p-AKT/AKT) were pronounced in A-ESCs from secretory phase (3.2-fold14inhibition versus control) than that from proliferation phase (2.3fold inhibition versus control).ConclusionMetformin inhibits cell growth via AMPK activation and subsequent inhibition of PI3K/AKT signaling in A-ESCs, particularly during the secretory phase, suggesting more effective of metformin on A-ESCs from secretory phase.